OP0091 EXPANSION OF MYELOPEROXIDASE (MPO)-SPECIFIC IgM B CELLS IN ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODY (ANCA)-ASSOCIATED VASCULITIS
Authors: C. Wortel, R. van de Wetering, H. van Dooren, E. M. Stork, A. M. Bakker, A. S. Brehler, T. Kissel, S. Reijm, G. Stoeken-Rijsbergen, K. van Schie, A. Rutgers, L. A. Trouw, T. Huizinga, Y. K. O. Teng, C. van Kooten, P. Heeringa, R. Toes, H. U. Scherer
Journal: Annals of the Rheumatic Diseases (2022)

Background ANCA-associated vasculitides (AAV) are characterized by recurrent, chronic small vessel inflammation and deleterious organ damage. The main targets of ANCA are myeloperoxidase (MPO) and proteinase 3 (PR3). ANCA, B cells and the complement system are crucial to AAV pathogenesis, as evidenced by the clinical benefit of B cell depletion with rituximab and, more recently, the C5a receptor antagonist avacopan. While ANCA in serum have been studied extensively, phenotypic and functional characteristics of the underlying B cell responses remain largely unknown. Objectives To develop a flow cytometry-based technique for identifying MPO-specific B cells in the circulation of MPO-positive AAV patients in order to characterize this B cell response and its potential contribution to disease pathogenesis. Methods Human neutrophil-derived MPO was conjugated to two different fluorochromes and used to identify MPO-specific B cells by flow cytometry. An antigen-specific staining protocol was developed and validated using MPO- and PR3-specific hybridoma cells. MPO-specific B cells were phenotypically characterized and isolated from the peripheral blood of AAV patients by fluorescence activated cell sorting (FACS) and cultured as single cells. MPO-specificity was confirmed by ELISA on culture supernatants. B cell receptor (BCR) sequences were obtained from MPO-positive clones by full length ARTISAN-PCR and Sanger sequencing. MPO-specific IgG and IgM monoclonal antibodies (mAb) were produced to validate specificity and to examine their ability to activate complement. Finally, MPO-positive AAV patient plasma and plasma depleted of IgG or IgM was tested in in vitro complement assays. Results The newly developed, differential antigen labelling approach successfully identified MPO-specific but not PR3-specific hybridoma cells. Subsequently, we detected MPO-specific B cells in the circulation of MPO-positive AAV patients at a frequency of up to 1:1000 B cells. FACS sorting and single cell culture yielded an enrichment of MPO-specificity of ~80%. Notably, the majority of isolated, MPO-specific B cells (60-95%) displayed an IgM memory phenotype, which corresponded to the presence of anti-MPO IgM in plasma. The remainder of the MPO-specific cells were mainly IgG memory B cells and few naive cells. BCR sequencing revealed a polyclonal IgM response with diverse V-gene usage, consisting of both germline and highly mutated clones. Generation of mAb (n=5) confirmed MPO specificity by inhibition ELISA for both germline and somatically mutated clones. Interestingly, anti-MPO IgM mAb showed a high capacity for complement factor deposition upon MPO binding. MPO-specific complement assays with IgG- and IgM-depleted patient plasma showed that anti-MPO IgM activated complement much more efficiently than anti-MPO IgG. Conclusion We demonstrate the direct ex-vivo identification, isolation and characterization of MPO-specific B cells in human AAV. Intriguingly, we observed a remarkable expansion of MPO-specific, IgM-expressing memory B cells in patients. This so far unrecognized, active IgM-compartment may be clinically relevant, as both mAb and plasma-derived polyclonal MPO-specific IgM strongly activated complement, a pathway thought to play a central role in AAV. Next to these novel insights into autoreactive B cell biology in AAV, our findings now provide new opportunities for studying auto-reactive B cell responses in different clinical phases of AAV, amongst which active disease, remission and (imminent) flares. Disclosure of Interests None declared.